A conserved biological response to double-stranded RNA (dsRNA) mediates resistance to parasitic nucleic acids, encoded by transposable elements and viruses. Cleavage of dsRNA into small interfering RNAs (siRNAs) triggers repression of genes related by sequence to the siRNAs. This silencing response is related by mechanism to an endogenous regulatory process that represses the expression of protein-coding genes. The latter process is guided by microRNAs (miRNAs), which are produced from genes within the genome. The long-term goals of our research are to understand the molecular mechanisms of siRNA and miRNA action, and to understand what aspects of cell activities regulate silencing by small RNAs. To this end, we have developed genetic, biochemical and cell biological methods in whole Drosophila and in cells to achieve our goals. In previous work, we conducted screens for Drosophila mutations that perturb siRNA- and miRNA-mediated repression. Genes that were identified from the screens are the basis for research aims in this proposal. Our aims are to: 1) determine how the nuclear Cdk9 kinase recruits a protein complex to co-transcriptionally cleave RNA transcripts into miRNAs, 2) determine how miRNA-protein complexes in the cytoplasm specifically repress the translation of certain mRNAs, 3) learn how these miRNA-protein complexes become associated with trafficking organelles in the cytoplasm, thereby leading to the inactivation of the miRNA-protein complexes, and 4) understand how the Dicer ribonuclease cleaves RNA from infecting viruses to generate antiviral siRNAs. The significance to health is manifold. The ability of small RNAs to regulate virus, transposon, and organismal gene expression suggests that basic understanding will reveal control mechanisms that influence many aspects of biology. It will also have an impact on viral infectious diseases and non-infectious diseases such as cancer. Moreover, small RNAs are used experimentally to probe gene function and are being commercially developed as disease therapeutics, making basic understanding of these molecules an important biomedical research goal.